Alexa fluor 488 conjugated anti mouse

Similar to immunohistochemistry, tissue epitopes were unmasked and subsequently exposed to UV light for 24 h to reduce autofluorescence. Sections were incubated with blocking buffer for 2 h and with primary antibodies (tau, Aβ and GFAP) for 72 h at 4 °C twice. In addition, for staining with HSP90AA1, HSP90AB1 and BAG3 antibodies, the sections were incubated with blocking buffer for 1 h and with the antibodies for 48 h or 24 h at 4 °C (for details, see Supplementary Materials Table S1).
Subsequently, the sections were incubated with Alexa Fluor 594-conjugated anti-rabbit, Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 647-conjugated anti-goat antibodies (1:200; Thermo Fisher, Waltham, Massachusetts, USA) for 2 h and then with 0.05% DAPI for 10 min at room temperature. Sections were mounted and coverslipped with PVA-DABCO.

Formalin-fixed paraffin-embedded mouse testis sections of 5 µm thickness were used for immunofluorescence (IF) staining. Sections were deparaffinized and antigens retrieved by heating in sodium citrate buffer. Primary autoantibodies were derived from the serum of O50 and adult normal control mice. Secondary antibody used was Alexa Fluor 488-conjugated anti-mouse (Thermo Fisher). Testis cross-sections on microscopic slides were then counterstained with Hoechst 33342 and observed under a fluorescent microscope (Axio Imager M2, Zeiss, Germany). β-Galactosidase activity, which is used as a biomarker of senescence, was detected using a staining kit (C0602) from Beyotime Technology (Shanghai, China) according to the manufacturer’s instructions. Collagen fibers were visualized with Masson’s trichrome stain using a kit (XY1427N) from Shanghai Xin Yu Biotechnology (China) according to the manufacturer’s instructions.

2-4hr old and 0.5-1.5hr old fertilized embryos were collected on grape juice agar plates, de-chorionated with 50% bleach, fixed using heptane/methanol and stained as described in (Horner et al. 2006 (link)). Ovaries from 3-5-day old females were dissected in Isolation Buffer (Page & Orr-Weaver 1997 (link)) and fixed using Fixation Buffer (Radford & McKim 2016 (link)), incubated with 5µg/ml RNaseA overnight (Horner et al. 2006 (link)), and stained with propidium iodide. Mouse anti-αtubulin (Sigma, St. Louis, MO, catalog #T5168) was used at a dilution of 1:400. Mouse anti-γtubulin (Boster Biological Technologies, Pleasanton, CA, catalog #MA1114) was reconstituted in 1ml of PBS and used at a dilution of 1:100. Mouse anti-DROP-1, which stains sperm tails (Karr 1991 (link)), (kindly provided by T. Karr at Kyoto University) was used at 1:800 as described in Krauchunas et al. (2012) (link). Alexa Fluor 488 conjugated anti-mouse (Thermo Fisher) was used at 1:200. Propidium iodide was used at 10 µg/ml. Samples were examined and images were generated using a Leica TCS SP2 confocal microscope at the Cornell Imaging Core.

PCL (M_n = 700,000–900,000), sodium orthovanadate, sodium fluoride, β-glycerophosphate, DOX (doxorubicin), and BrdU were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pimonidazole hydrochloride was obtained from Hypoxyprobe Inc. (Burlington, MA, USA). The following antibodies were used for the study: Alexa Fluor 594 phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), anti-HIF-1α (R&D Systems, Minneapolis, MN, USA), anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA), anti-F-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-BrdU (Thermo Fisher Scientific, Waltham, MA, USA), anti-pimonidazole (Hypoxyprobe Inc.), anti-Ki67 (Cell Signaling Technology, Beverly, MA, USA), and cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA). The following secondary antibodies were used: Alexa Fluor 488-conjugated anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 594-conjugated anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 594-conjugated anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA), and HRP-conjugated anti-mouse (Cell Signaling Technology, Beverly, MA, USA).

To confirm the quality of the prepared tubular liver tissue, paraffin-embedded tissue sections were prepared for observation. The tissues were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical) for 6 h at 4 °C, dehydrated with ethanol, permeabilized with Lemosol (FUJIFILM Wako Pure Chemical), and embedded in paraffin. Then, sections of 5 μm thickness were prepared. After deparaffinization by Lemosol and ethanol, the sections were subjected to staining with hematoxylin and eosin (HE) and immunostaining. For immunohistochemistry (IHC), after heat-induced epitope retrieval (HIER) using citrate buffer (pH 6), the sections were incubated with the primary antibody for CD31 (1:100, ab28364, Abcam PLC, Cambridge, UK), followed by a secondary antibody (ImmPRESS Reagent, Anti-Rabbit Ig, Vector Laboratories, Inc., Burlingame, CA, USA). Finally, the sections were colorized with DAB (ImmPACT DAB, Vector Laboratories) and counterstained with hematoxylin. For immunofluorescence, after HIER, the sections were incubated with the primary antibodies against CD31 and CK18 (1:200, MA5-12104, Thermo Fisher Scientific Inc., Waltham, MA, USA) and secondary antibodies (1:200, Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit, Thermo Fisher Scientific Inc.).

Immunofluorescence analyses were performed as described elsewhere [43 (link), 44 (link)]. Briefly, mice at indicated age were deeply anesthetized, and transcardially perfused with phosphate buffered saline (PBS) then 4%(w/v) paraformaldehyde in 0.1 M phosphate buffer for 10 min, respectively. After the incubation with 30%(w/v) sucrose in PBS, the dissected lumbar spinal cords were embedded in Tissue-Tek OCT compound medium (Sakura Finetek, Tokyo, Japan), and froZen at − 80 °C until use. After blocking, the 12 μm-sliced spinal cord sections were incubated with primary antibodies for overnight at 4 °C. Bound primary antibodies were detected with Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 546-conjugated anti-goat IgG secondary antibodies (both used in 1:1000) (Thermo Fisher Scientific Inc., Waltham, MA, USA). Immunofluorescence images were obtained by a confocal laser scanning microscopy (LSM-700; Carl Zeiss AG, Oberkochen, Germany) and the equipped software (Zen; Carl Zeiss AG). Cholinergic large synaptic terminals on α-motor neurons (C-boutons) were identified as contacting sites of ChAT and Kv2.1 on the surface of ChAT-positive motor neuron soma in ventral lumber spinal cords. For quantification, more than 50 motor neurons in three animals per genotype were counted for C-boutons based on the immunofluorescence images obtained by confocal laser scanning microscopy.

Rabbit polyclonal antibody against BiP (abcom; ab21685), rabbit polyclonal antibody against β-Tubulin III (Sigma-Aldrich; T2200), Amersham ECL rabbit IgG, HRP-linked whole Ab from donkey (GE Healthcare Life Sciences; NA934) were used for Western blotting. Mouse monoclonal antibody against KDEL (10C3) (Enzo Life Sciences; ADI-SPA-827) and Alexa Fluor 488-conjugated anti-mouse (Thermo Fisher Scientific; A-11017) and Alexa Fluor 568-conjugated anti-rabbit IgG (Thermo Fisher Scientific; A-21069) were used for immunofluorescent staining. Rabbit polyclonal antibody against FKBP22/FKBP14 (15884-1-AP; proteintech) was used for both Western blotting and immunofluorescent staining.